2-Deoxy-D-Glucose: A Novel Pharmacological Agent for Killing Hypoxic Tumor Cells, Oxygen Dependence-Lowering in Covid-19, and Other Pharmacological Activities

The nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) has shown promising pharmacological activities, including inhibition of cancerous cell growth and N-glycosylation. It has been used as a glycolysis inhibitor and as a potential energy restriction mimetic agent, inhibiting pathogen-associated molecular patterns. Radioisotope derivatives of 2-DG have applications as tracers. Recently, 2-DG has been used as an anti-COVID-19 drug to lower the need for supplemental oxygen. In the present review, various pharmaceutical properties of 2-DG are discussed.


Introduction
2-Deoxy-D-glucose (2-DG, 2-deoxy-D-arabino-hexopyranose) is a natural [1], nonmetabolizable glucose analog and a competitive inhibitor of glycolysis [2] in which the 2hydroxyl group is replaced by hydrogen ( Figure 1). 2-DG blocks the activity of diferent enzymes involved in glycolysis, leading to cell death. Hyperglycemic condition aggravates cancer cell proliferation, infammatory conditions, and viral infection [3]. In this review, the pharmaceutical properties of 2-DG have been discussed. Tis review article describes the pharmacological properties specifc to 2-DG and its isotopic derivatives but excludes substituted derivatives of 2-DG.

Toxicity of 2-DG.
Toxic efects of 2-DG result from its ability to block glycosylation [4,5] but not glycolysis [6]. Ketogenic Diet increases tolerance against glycolysis inhibitors [7]. Experimental results show that 2-DG is a relatively harmless compound at low doses, but that it can lower blood pressure and slow breathing at higher doses [8].
Most of the studies indicate that a clinically tolerable dose of 2-DG is up to 63 mg/kg/day [9]. Beyond this several side efects are observed including reversible hyperglycemia, gastrointestinal bleeding, and QTc prolongation [9]. However, recent studies carried out by DRDO use a higher dose of 90 mg/kg/day [10]. Exposure to 2-DG causes cytotoxicity and radiosensitization via a mechanism involving changes in thiol metabolism and these efects may be more prominent in transformed vs. normal cells [11].
precision suitable for the analysis of active pharmaceutical ingredients and drug products [12]. Te method is suitable for the standardization and quality control of APIs and drugs [12]. UV-HPLC (195 nm) has been used to detect and quantify 2-DG using a μBondapak 10 μm NH 2 column and a Varian MicroPak 10 μm NH 2 column. Te retention time is usually four minutes with an eluent 85% MeCN/H 2 O [13]. Polymer-based amino column (HILICpak VG-50 4E column) and Shodex SUGAR SC1011 columns have also been used to separate 2-DG and glucose. Pharmacokinetic studies of 2-DG involve the estimation of 2-deoxyglucose in the plasma [14]. For this purpose, precolumn fuorescent derivatization was achieved by reductive amination of 2-DG using sodium cyanoborohydride and 2-aminobenzoic acid [14].

Pharmaceutical Profile of 2-DG
Te molecule 2-DG follows Lipinski's rule of fve and has several activities, kills hypoxic tumor cells, and lowers oxygen dependency in case of Covid-19 ( Figure 2). A number of studies have described diferent biological activities [3], but it is not approved as a drug until May 2021 [15]. In May 2021, 2-DG was found an emergency use as an anti-Covid-19 drug allowing patients to recuperate more quickly by lowering the need for supplemental oxygen. 2-Deoxyglucose (2-DG) is a toxic glucose analog. 2-DG has a pleiotropic mechanism of action ( Figure 3) [16][17][18].
2-DG mimics mannose has brought up the prospect of developing it as an antiviral agent, in addition to restricting cancer growth [21]. Glycolytic inhibitors such as 2-DG potentiate the activity of Paclitaxel [22].

Inhibition of Infammation.
Recent reports suggest the usefulness of 2-deoxy-D-glucose in the inhibition of infammation [23], controlling respiratory infections, and treatment of human genital herpes [24]. 2-DG shows antiinfammatory activities through the regulation of antiinfammatory mediators and the polarization of macrophages [3]. During ocular infection with herpes simplex virus (HSV), stromal keratitis occurs due to infammatory reactions in the eye. In vivo experiments to limit glucose utilization using 2-DG showed diminished lesions with fewer proinfammatory efectors [25]. 2-DG also acts as a potential energy restriction mimetic agent (ERMA) [26], and has application in reducing acute infammation events caused by pathogen-associated molecular patterns (PAMPs) [26], 2-DG application as ERMA in drinking water can help to avoid pathogenic exposure-induced infammatory events, which can help to prevent both acute and chronic infammatory illnesses [26]. In the mice model, treatment with 2-DG (0.4% w/v in drinking water) reduces infltration of infammatory cells, infammatory signaling activation, oxidative stress, capillary damage in lungs [26], and reduced the BALF and serum [26]. 2-DG reduces the infammatory responses triggered by glycogen accumulation caused by coal dust because macrophages have reconstituted glycogen metabolism [27]. A study on dextran sulfate sodium-induced colitis-mouse (mouse model for infammatory bowel disease), alleviated laminarin-induced arthritis in the SKG mouse (model for human rheumatoid arthritis), LPS shock (model for a cytokine storm), and LPS-induced pulmonary infammation (model for COVID -19) suggested that 2-DG is efective for the treatment of various infammatory disease due to its capability to inhibit cytokine receptor glycosylation [28].      Advances in Pharmacological and Pharmaceutical Sciences accumulation of 2-DG-6-P in cells inhibits phosphoglucose isomerase (PGI) activity. Tus, 2-DG limits glucose uptake and the downstream metabolic pathway, which depletes the ATP level and induces cell death. Several applications of 2-DG are known for the efcient elimination of cancer cells, and the synergetic efect of 2-DG has been reported in the literature [29]. Te limited therapeutic efect of 2-DG in cancer treatment is overcome by its benefcial synergistic anticancer efect with other therapeutic agents or radiotherapy [20] by blocking glycolysis in hypoxic tumor cells and subsequent cell death [30]. 2-DG manipulates its similarity to glucose and the tendency of cancer cells to utilize glycolysis even in the presence of oxygen, a process known as aerobic glycolysis or the Warburg efect [31,32]. 2-DG acts as a D-glucose mimic, suppressing glycolysis by forming and accumulating 2-deoxy-D-glucose-6-phosphate (2-DG6P) inside cells, blocking hexokinase and glucose-6-phosphate isomerase, and causing cell death ( Figure 2) [33].
Nanoparticles or nanosized molecules show the pharmaceutical efect diferently. Nanoliposomes have been used in various drug delivery systems. 2-DG-containing nanoliposomes have shown inhibition of glycolysis in cancer cells. Te synergistic efect of these 2-DG-loaded liposomes with the coloaded drug enhances mitochondrial depolarization and subsequent apoptosis [34].
Studies have shown that 2-Fluoro-Deoxyglucose resembles glucose more closely than 2-DG. Lampidis employed a QSAR-like method combined with a fexible coupling strategy to determine that the analog binding affnities to hexokinase I decrease as the size of the halogen increases [30].
D-glucose had the highest afnity binding afnity to hexokinase I, followed by 2-FG and 2-DG [30]. 2-DG dramatically increased ATP depletion and 4E-BP1 phosphorylation. On the other hand, high amounts of 2-FDG can prevent further protein glycosylation processes by competing with glucose [35].
Several drugs have been tested in combination with 2-DG to inhibit the growth of cancer cells. Tese drugs and corresponding cancer types are listed in Table 1.

Autosomal Dominant Polycystic Kidney Disease (ADPKD).
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by defective glucose metabolism. Chiaravalli studied the efect of low doses of 2-DG on ADPKD progression in orthologous and slowly progressive murine models created postnatally by inducible inactivation of the Pkd1 gene. Tese studies established proof-ofprinciple support for the use of 2-DG as a therapeutic strategy in ADPKD [72]. 2-DG can suppress the activity of seizures and retards the epilepsy progression in vitro as well as in vivo [73].
Te combination of metformin and a low dose of 2deoxyglucose synergically inhibits cyst formation and human polycystic kidney cell proliferation [74,75]. Cheong and his colleagues reported that 2-DG with metformin can prevent tumor growth in mouse xenograft models [76].
Due to its similarity in the structure of glucose and nonparticipation in glycolysis [77], 2-DG has emerged as a tool for metabolism-independent GS investigations [78,79]. Studies on the efect of 2-DG treatment on the mHypoE-29/1 cell line suggest that the metabolismdependent GS pathway is responsible for glucose detection in neuronal cells and the downregulation of AgRP mRNA levels.
Te efect of exposure to 2-DG on AgRP mRNA levels in the adult mHypoA-NPY/GFP model and the embryonic model showed that both cell lines expressed Tas1R2 and Tas1R3 mRNA transcripts, indicating the involvement of metabolism-independent glucose sensing mechanisms. Tese studies also suggest that control of AgRP mRNA expression in embryonic cells is metabolism-dependent, whereas adult cells may act in a metabolism-independent manner [80].
2-DG inhibits T-cell-mediated cytolysis since 2-DG metabolites compete with glucose metabolites for key enzymes (such as glycosyltransferases) that are necessary for cytolysis expression [83].
Te synthesis and characterization of CyNE 2-DG, a new NIR fuorescent DG analog, was reported by Vendrell et al. Te coupling of 2-deoxy-glucosamine and tricarbocyanine carboxylic acid in the presence of the coupling reagent, i.e., HATU results in the formation of CyNE 2-DG [84].
Immobilization and 2-DG-induced central neuroglycopenia should be identifed as diferent types of stressful stimuli, causing their efects through diferent neural pathways, based on secretory, hemodynamic, and synthesis of adrenal catecholamine rate responses [85].
Te suppression of proteoglycan production by the GAG chain could be linked to ATP depletion in cells. In confuent primate VSMCs, the ATP content of cells decreased by 25-30% after exposure to 2-deoxyglucose. ATP levels and proteoglycan synthesis recovered to baseline after 2-DG was removed [86]. 2-DG inhibits substance P (SP) production in the bodies of sensory ganglion cells of vagal cells, as well as its bidirectional transit to the CNS, thoracic, and abdominal viscera [87]. Total protein synthesis was not afected when the ratio of hexose to 2-DG was 20 1 or greater. Under similar conditions, invertase and acid phosphatase production and secretion are inhibited by 2-DG. Glucan formation was also inhibited. Te mechanism of inhibition of total uptake of external sugar by 2-DG after a lag period Advances in Pharmacological and Pharmaceutical Sciences involves intracellular 2-DG-6-P, which directly inhibits the conversion of fructose-6-P to glucose-6-P and mannose-6-P by phosphohexose isomerases; simultaneously decreasing the transport of fructose or maltose into cells [88][89][90]. 2-DG inhibits cellular repair phenomena, even after completing the unscheduled DNA synthesis [91][92][93][94]. 2-DG inhibits DNA repair and the repair of potentially lethal damage in cancerous cells. Tese two phenomena were studied in respiratory-defcient yeast cells irradiated with X assayed by unscheduled DNA synthesis and cell viability after irradiation, respectively [92][93][94].
LPS-induced aerobic glycolysis is inhibited by 2-DG; therefore, collagen synthesis is also inhibited [95]. Glycolysis suppression results in decreased muscle protein synthesis as a result of decreased basal mTORC1 signaling [96].

Antiseizure Efects and Retarding Efect in Epilepsy
Progression. 2-DG exhibits antiseizure efects through the netrin-G1-KATP signaling pathway by upregulating K (ATP) subunits kir6.1 and kir6.2 [97,98]. In animal models, it retards the progression of epilepsy [99]. Te epileptic brain shows dynamic metabolic changes. Focal zones of onset of seizures are hypometabolic during the interictal period and hypermetabolic during seizures [100]. Terefore, glycolysis plays an important role in these dynamic metabolic changes; therefore, 2-DG could abrogate seizure activity and retard epilepsy progression. [101]. Later it was found that 2-DG inhibits the propagation of epidemic diarrhea virus [102] in human rhinoviruses [103], pandemic SARS-CoV-2 [103,104], and endemic human coronaviruses [103]. Tese in vitro studies suggested broad-spectrum antiviral use of 2-DG [103]. 2-DG limits viral proliferation in the body by selectively killing cells infected with the SARS-CoV-2 virus by halting energy production and viral synthesis [104]. 2-DG has limited use in SARS-CoV-2 patients sufering from stroke, hypoxicischemic encephalopathy, and other critical illnesses [105][106][107]. In Phase II clinical trials, the addition of 90 mg/ kg/day of 2-DG to the standard of care (SOC) for the treatment of moderate to severe COVID-19 demonstrated clinical beneft over SOC alone [107].

COVID-19 and 2-DG. Perris hypothesized the use of modifed sugars for the treatment of viral infections in 2007
Te Drugs Controller General of India (DCGI) has approved the emergency use of 2-deoxy-D-glucose (2-DG, 1) on 1 May 2021 [15]. 2-DG is used as an adjunct therapy in patients with mild to serious COVID-19 to recover quickly by reducing the supplemental oxygen requirement.

Docking and Computational Studies. COVID-19
disease has emerged as an epidemic of the 21st century due to the nonavailability of efective antiviral agents, as well as its pathophysiology [108]. Te fragment molecular orbital (FMO) method has been used to characterize SARS-CoV-2 S-protein binding interactions with the ACE2 and B38 Fab antibodies involved in ACE2-inhibitory binding. Tese studies helped to understand the amino acid residues critical for molecular recognition between the S-protein and the ACE2 or B38 Fab antibody [109]. Te binding of 2-DG and 1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucose to viral main protease 3CLpro and NSP15 endoribonuclease is studied using molecular coupling techniques. Tese studies show that viral receptors are inactivated due to the formation of a hydrogen bond between 2-DG and proline residues. Furthermore, 1,3,4,6-Tetra-O-acetyl-2-deoxy-D-glucose forms a hydrogen bond with the glutamine amino acid residues of the viral spike glycoprotein [110]. Te coronavirus disease 2019 (COVID-19) pandemic has highlighted the value of FDG-PET/CT in diagnosis [111,112]. Molecular coupling and molecular dynamics simulations showed that 2-DG had a positive interaction with SARS-CoV NSP12 and the SARS-CoV-2 RBD spike-ACE2 complex [113].
Drug-drug interactions (DDI) and synergistic regulatory potential have been investigated in a report describing a molecular coupling and simultaneous molecular dynamics simulation of multiligands to study the combined efect of 2-DG with other 62 selected drugs and phytochemicals. In terms of binding energy, the combination of 2-DG with Ruxolitinib, Telmisartan, and Punicalagin was superior to that of the selected individual compound [114].

Similarity between the Pathophysiology of Cancer Cells and SARS-CoV-2 Infected Cells with Respect to Glycolysis Inhibitors. Cancer cells and virus-infected cells have simi-
larities; both require a large amount of energy because of their very high proliferation rate. Tis high energy requirement increases glucose uptake by infected cells and uses glycolysis and glycosylation for energy production [115]. It promotes mitochondrial signaling, i.e. aerobic glycolysis. Hyperglycemic conditions, such as diabetes, facilitate the invasion and propagation of SARS-CoV-2 and an aggravated immune response [116,117]. Tus, impaired glucose metabolism will destroy infected cells and viruses in parallel with cancerous cells. Infected cells hungry for energy will absorb a high amount of glucose antimetabolite compared to normal cells. Tese facts became the basis of the use of 2-DG in the treatment of COVID-19 [106,118]. . COVID-19 is a serious acute respiratory disease associated with cardiovascular complications. Te interaction of virus Nsp6 with host proteins from the MGA/ MAX complex (MGA, PCGF6, and TFDP1) was studied by expressing the SARS-CoV-2 protein in the hearts of Drosophila and using transcriptomic data. Tis interaction blocks the antagonistic MGA/MAX complex, which shifts the balance to MYC/MAX and activates glycolysis. Nsp6mediated upregulation of glycolysis disrupts cardiac mitochondrial function, which increases ROS in heart failure. Tis could explain the cardiac pathology associated with COVID-19. Inhibition of glycolysis with 2-deoxy-D-glucose reduces the Nsp6-induced cardiac phenotype in fies and mice. Tese fndings suggest glycolysis as a pharmacological target for COVID-19-related heart failure [119]. 6 Advances in Pharmacological and Pharmaceutical Sciences Tirumalaisamy reported Hyaluronic acid-2-DG conjugated as a novel drug in the treatment of COVID-19 [120]. Replication of SARS-CoV-2 in Caco-2 cells was prevented by inhibiting glycolysis and nontoxic 2-DG concentrations [121]. SARS-CoV-2 replication requires high energy and is supported in colon cancer cells by increased carbon metabolism. Tus, glycolysis inhibitor 2-DG inhibits SARS-CoV-2 replication [121].

Resistance to 2-DG.
Overexpression of the odr1 gene of Schizosaccharomyces pombe produces a strong resistance to 2-DG with diferent resistance mechanisms for budding yeast and fssion yeast [122]. S. pombe Odr1 hydrolase can act in the toxic form of 2-DG, similar to Saccharomyces cerevisiae Dog1/Dog2, which encodes HAD-like hydrolase enzymes and exhibits specifc 2-DG-6-phosphatase activity [122].

Other
Applications. Te use of 2-DG with low-dose radiation therapy is also suggested for anticancer, antiinfammatory, and antibacterial/antiviral efects [123]. 2-DG is also used as a hypoglycemic stimulus [66]. 2-DG has been used to assess glucose uptake activity by measuring the intracellular accumulation of 2-DG [124]. [ 14 C]deoxyglucose ([ 14 C]-2-DG) is used as a glucose tracer. Te method has been used to trace the exchange of glucose between plasma and brain and its phosphorylation during hexokinase glycolysis in tissues [125]. Direct evaluation of local cerebral metabolic activity is possible by using [ 14 C]-2-DG in the quantitative autoradiographic method. Tis method provides access to quantitative measurement of glucose utilization and histological identifcation of afected cortical areas [126].
PET is used to measure glucose metabolism using the 2-DG method proposed by Sokolof et al. Similarly to 2-DG, F18-labeled deoxyglucose is also an analog of glucose and is taken up in the brain and is phosphorylated in glycolysis. Te absence of a C-2 hydroxyl prevents glycolysis; thus, it builds up intracellularly.
PET measures arterial tracer concentration, glucose uptake kinetics, and the regional cerebral glucose metabolic rate (rCMRGlc) can be measured or calculated. Positron computed tomography (PCT) and (F-18)2-fuoro-2-deoxy-D-glucose (FDG) are used to measure the local cerebral metabolic rate of glucose [127,128]. Te PCT method is used to measure glucose uptake in breast cancer cells [129]. Te method is also used for the study of brain cells [130], lung cells [131], upper airway infammation [132], infection in the case of multiple ventriculoperitoneal shunts [133], and myocarditis-STREAM [134].
Glucal is the glycal formed from glucose and is one of the common starting materials for the synthesis of 2-DG. A general conversion involves the bromination (or halogenation) of Glycal at C-2 followed by the replacement of bromine with hydrogen. Bromination takes place in nucleophilic solvent using molecular bromine. Binkley et al. reported photolysis of α and β anomers of 7 to yield α and β anomers of 8. Treatment of 8 with Baker ANGA-542 ion exchange resin in methanol produced 2-DG with a yield of 78% yield [141]. Compound 7 was synthesized by nucleophilic bromination of 4 followed by hydrolysis and acetylation ( Figure 5) [141].
Masuda and coworkers reported the synthesis of 2-DG from D-glucose. 2-Deoxy-D-glucose was prepared in three steps from natural D-glucose dispensing with any protection/deprotection procedure and was obtained in 48% yield ( Figure 6) [146].

Future Perspectives
2-DG has been investigated for combination therapy to inhibit cancerous cells. However, the recent use of 2-DG to treat COVID-19 patients under emergency conditions has opened up new hope for the development of new antiviral medicines. COVID-19 is a viral disease caused by SARS-CoV-2 variants. Mutation in the virus is of much concern, which results in the deactivation of available drugs and monoclonal vaccines. Tus, a target that is not directly affected via mutation has its own value. Targeting glycolysis in energy-hungry infected cells will stop the multiplication of the virus [117,119,151]. Further, all variants of SARS-CoV-2 follow rewired glycolysis, which makes 2-DG as a good Advances in Pharmacological and Pharmaceutical Sciences starting point for the development of broad-spectrum antiviral [103]. Normal cells could rely on the citric acid cycle and oxidative phosphorylation using Acetyl-CoA from Fatty acids and ketone bodies. Te application of 2-DG is dosedependent, thus safe doses should be determined by their efects.

8
Advances in Pharmacological and Pharmaceutical Sciences

Conclusions
2-DG is a dual D-glucose and D-mannose mimetic and exploits increased glucose metabolism to kill glycolytic cells. Te biological efects of the molecule 2-DG include inhibition of sugar uptake, inhibition of glucose metabolism, antiviral, anti-infammatory, anticancer, antiepileptic activity, and more. It has found use as a tracer, cancer diagnostic tool, and metabolite inhibitor. Positron emission tomography has made use of 18F-fuoro-2-deoxy-D-glucose. Due to its ability to reduce infammation and kill glycolytic cells, 2-DG exhibits activity against SARS-CoV-2. Antiviral properties of 2-DG have paved the way for the development of new antiviral drugs and therapies for hyperglycemic patients. Further investigation is necessary to determine the safest doses for various applications, the mechanism of action, the toxicity, and the interactions of 2-DG with various edibles.

Data Availability
Data sharing is not applicable to this article.

Conflicts of Interest
Te authors declare no conficts of interest.  Advances in Pharmacological and Pharmaceutical Sciences 9